Around 147 base pairs of eukaryotic DNA are wrapped around core histone proteins into a nucleosome. The nucleosome is a highly stable structure which forms the basic repeating subunit of chromatin. During chromatin replication in S phase, parental nucleosomes are disrupted ahead of the replication machinery and are segregated or “recycled” onto both daughter DNA strands following replisome progression. To maintain nucleosome occupancy and genome integrity, newly synthesised histones are assembled into nascent DNA during replication-coupled de novo nucleosome assembly. Several components of the replisome and different histone chaperones have been implicated in both pathways of replication-coupled nucleosome assembly. To differentiate the roles of these factors, I have leveraged an in vitro reconstituted chromatin replication system using purified proteins from S. cerevisiae.
In this study, I have established that no additional histone chaperones, such as FACT, are required for replisome progression through chromatin or for parental histone recycling. During in vitro chromatin replication, parental histones are assembled into full nucleosomes and sub-nucleosomal complexes in a replication-coupled manner. However, when I abrogated all of the known histone-binding sites within the replisome, chromatin replication and parental histone recycling were not perturbed. Therefore, these data support the hypothesis that there is a fundamental DNA-to-DNA mediated mechanism of parental histone transfer during chromatin replication.
In vitro reconstituted replication-coupled de novo nucleosome assembly requires CAF1; however, CAF1 is not essential in S. cerevisiae. Here, I further elucidate this CAF1-mediated mechanism of nucleosome assembly and explore the role of the histone chaperone FACT in the process. I describe that FACT alone does not assemble nucleosomes in a replication-coupled manner but present evidence that FACT directly interacts with CAF1 in vitro. These data lead to the hypothesis that FACT may cooperate with different factors to facilitate replication-coupled de novo nucleosome assembly.